A strain of Streptococcus faecalis var. zymogenes, designated JH1, had highlevel resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chlioride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 106 and 38 x 106. An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 106 was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10.. Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 106 molecular weight plasmid but lost the 6 x 106 molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 106 molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 106 remnant which was incompatible with the complete replicon.

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A sequence of deoxyribonucleic acid of 2.7 times 10-6 to 3.3 times 10-6 daltons which includes the TEM beta-lactamase gene is present on the small plasmid RSF 1030 (R-Amp). This same sequence is present on plasmid derivatives that have received a translocation of deoxyribonucleic acid specifying the TEM beta-lactamase and is also present on naturally occurring plasmids of the F1, F11, N, X, O, I, C, and W incompatibility groups that do not specify ampicillin resistance or specify O-type beta-lactamases.

Genes conferring resistance to aminoglycoside-aminocyclitol antibiotics in three group D streptococcal strains, Streptococcus faecalis JH1 and JH6 and S. faecium JH7, and to chloramphenicol in JH6 are carried by plasmids that can transfer to other S faecalis celLs. The aminoglycoside resistance is mediated by constitutively synthesized phosphotransferase enzymes that have substrate profiles very similar to those of aminoglycoside phosphotransferases found in gram-negative bacteria. Phosphorylation probably occurs at the aminoglycoside 3'-hydroxyl group. Plasmid-borne streptomycin resistance is due to production of the enzyme streptomycin adenylyltransferase, which, as in staphylococci and in contrast to that detected in gram-negative bacteria, is less effective against spectinomycin as substrate. Resistance to chloramphenicol is by enzymatic acetylation. The chloramphenicol acetyltransferase is inducible and bears a close resemblance to the type D chloramphenicol acetyltransferase variant from staphylococci.In recent years it has become clear that many of the genes conferring resistance to antibiotics in group D streptococci are plasmid borne (4,5,13). The plaids can transfer between group D streptococcal strains; some plasmids are conjugative (13), and nonconjugative plasmids can be mobilized by conjugative plasmids (9).In gram-negative bacteria and Staphylococcus the mechanism of plasmid-mediated resistance involves the production of enzymes that inactivate the antibiotic by either modification or destruction of the antibiotic structure (8). This is in contrast to chromosomally detennined antibiotic resistance, which is more usually due either to prevention of entry of the antibiotic into the cell or to a specific alteration of the antibiotic target site (8). In this report it is shown that, like the plasmid-borne resistance in the gram-negative bacteria and staphylococci, plasmid-linked resistances to aminoglycoside-aminocyclitol antibiotics and chloramphenicol in group D streptococci are also mediated by enzymatic modification ofthe antibiotic. The properties of the Streptococcus faecalis enzymes are compared with the similar enzymes of staphylococci and the gram-negative bacteria. (12,13). Derivative strains were isolated by selection of spontaneous mutants (13).Media. Brain heart infusion broth and agar (Oxoid) were used, except where stated. All incubations were at 370C.MIC determinations. The method of Steers et al. (18) was used to determine antibiotic minimal inhibitory concentrations (MICs).Radiolabeling, isolation, and characterization of plasmid DNA. Plasmid DNA was isolated by cesium chloride-ethidium bromide density gradient ultracentrifugation and characterized by neutral sucrose gradient analysis as described previously (12, 13).Calculation of plasmid molecular weight. The empirical formulae of Barth and Grinter (1) were used to calculate plasmid molecular weights.Transfer of antibiotic resistance traits. The technique described by Jacob and Hobbs (13) was used in transferring antibiotic resist...

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Bcn-), and those from strain JH3 had also